Human respiratory syncytial (RS) virus, an enveloped non-segmented negative strand RNA virus, is the major etiologic agent of severe lower respiratory illness of infants and young children. We have used recombinant DNA techniques and biochemical analysis to explore the genome of this virus. A cDNA library representing the poly(A) containing RNA's from RS virus infected cells was constructed in E. coli (HB101) using plasmid pBR322 as a vector. The strategy for cloning avoided the traditional approach that includes a self priming reaction for second strand synthesis and subsequent S1 nuclease treatment to remove the resulting hairpin. We have determined the complete DNA sequences of six RS viral genes cloned in recombinant plasmids. A seventh gene that appears to code for the fusion protein, has also been sequenced; this recombinant lacks about 70 nucleotides corresponding to the 5' end of the message. There was conservation of a PuGGGCAAAT . . . . sequence at the 5' end of the 7 RS viral transcripts. This consensus initiation sequence differs from that found at the 5' end of paramyxovirus and rhabdovirus transcripts. The initiating nucleotide is probably G in all RS virus genes.